All folate-dependent one-carbon transfer reactions had been considered active only with the naturally occurring isomers of tetrahydrofolate (H4folate). Recently, however, in vitro and in vivo experimental evidence suggests that 10-formyldihydrofolate (10-HCO-H2folate) has physiological roles. A hypothesis is proposed that a significant portion of the carbon-2 (C2) of the purine ring originates from the formyl group of 10-HCO-H2folate. The following specific aim is proposed to test the hypothesis. To identify the folate origin of the C2 and C8 positions of the purine ring, in vivo competition experiments will be performed. Subjects will be given an oral dose of 13C-sodium formate and subsequently a dose of one of the following five formylfolates (containing a one-12C moiety); natural and unnatural isomers of 5-formyltetrahydrofolate and 5,10-methenyltetrahydrofolate and 10-HCO-H2folate. Following the oral doses of 13C-sodium formate and a formylfolate, urine samples will be collected for three days, and the 13C-enrichment pattern at the C2 and C8 positions of uric acid will be determined by mass spectrometry. At the beginning of this three-day period, an in vivo 13C-formylfolate pool will be established. The formyl carbon of 13C-formylfolates will compete with that of unlabeled formylfolates (given in a large dose) for the incorporation into the C2 and C8 positions of the purine ring. 10-HCO-H2folate will have the strongest negative effect on 13C enrichment at the C2 position and no effect on the 13C enrichment at the C8 position, since this folate is a better substrate for AICAR transformylase than is natural 10-formyltetrahydrofolate. The study will provide basic knowledge of a new folate pathway involving 10-HCO-H2folate in humans. If this pathway is proven, derivatives of 10-HCO-H2folate could be used to inhibit de novo purine biosynthesis for developing new anticancer drugs. [unreadable] [unreadable]